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Influencing Notch signalling in virus associated tumours
Bujnovská, Ludmila ; Šmahelová, Jana (advisor) ; Grantz Šašková, Klára (referee)
The Notch signalling pathway affects cell differentiation, apoptosis and proliferation. It is an evolutionarily conserved signalling pathway important during embryogenesis and ontogenesis. Its deregulation can lead to carcinogenesis. Cells of various tumour types often contain gene mutations or other abnormalities in the Notch pathway. Its function with regard to oncogenesis has a dual character - in some cases it acts as an oncogenic pathway, in others it has a tumour-suppressive effect. This depends on the cellular context. A large group of tumours with proven abnormalities in the Notch signalling are head and neck squamous cell carcinomas (HNSCC). It is the 7th most common tumour type and the lethality rate is high. Almost 25 % of these tumours are etiologically related to human papillomavirus (HPV) infection. The cell cycle signalling pathways, including the Notch pathway, are the primary target of oncogenic viruses where HPV are no exception. This thesis describes changes in the Notch signalling pathway in HPV-induced HNSCC and the effect of viral oncoproteins E6 and E7 on this signalling pathway.
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Characterizing DDI2 protein interaction by solution NMR
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR
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Effect of polyhistidine modification of viral particles on their intracellular localization and gene delivery to the nucleus
Číhařová, Barbora ; Španielová, Hana (advisor) ; Grantz Šašková, Klára (referee)
Viral vectors derived from mouse polyomavirus are a convenient tool for studying the targeted delivery of therapeutical agents into the cells and cellular organelles. Vectors derived from mouse polyomavirus face difficulties similar to other nanoparticles, as they often end up trapped inside an endosome where they are subsequently degraded. This diploma explored the potential of vector modifications, which have the potential to make the transport to the nucleus or cytosol more effective. This work had particularly focused on increasing the transduction efficiency by modifying particle's internally localized VP3 capsid protein with covalently bound membrane-penetrating peptides. Primary covalent genetic modification to the VP3 protein was the polyhistidine peptide KH27K. Its potential of improving the transduction effectivity was compared with two other peptide modifications - LAH4 and R8. The results of the transduction test showed that covalently bound R8 peptide had many-fold improved the transport to the nucleus when compared to the unmodified particles. The modification with LAH4 peptide had been regarded more effective only when was associated with the particles non-covalently. In such scenario the transduction efficiency rose 40-times when compared with unmodified particles. Polyhistidine...
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Preparation of nanoparticles for hepatitis B viral therapy
Kružíková, Zuzana ; Grantz Šašková, Klára (advisor) ; Žáčková Suchanová, Jiřina (referee)
Hepatitis B virus (HBV) represents one of the hot topics of current basic and pharmaceutical research. Although an effective vaccine against HBV exists since 1982, the world prevalence of chronic infection is still alarming. The infection can lead to significant liver damage, often resulting in hepatocellular carcinoma. Chronic HBV infection cannot be cured due to the fact that the viral genome persists in the infected hepatocyte hidden from the host immune response as well as from the antiviral treatment. One of the novel approaches aiming for HBV cure suggests that this cccDNA pool could be destroyed using gene editing tools such as CRISPR/Cas9 system. In order to shift this gene editing system to possible medicinal application, CRISPR/Cas9 has to be specifically delivered into the target cell in order to minimize its putative off-target activity. This thesis focuses at first on the design and efficacy testing of new sgRNAs targeting HBV cccDNA and secondly, it describes modular lipid nanoparticles developed specially for delivery of the CRISPR/Cas9 system in the form of RNA. Keywords: hepatitis B virus, CRISPR/Cas9, gene editing, lipid nanoparticles, mRNA delivery, targeted delivery
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Characterizing DDI2 protein interaction by solution NMR
Staníček, Jakub ; Grantz Šašková, Klára (advisor) ; Obšil, Tomáš (referee)
Human DDI2 protein is a dimeric aspartic protease that has been recently found to play an important role in DNA damage repair and transcriptional regulation of the proteasome expression. Current insights into the mechanistic details of both functions are still quite limited. We have previously identified the human RAD23B protein to interact with the DDI2 protein. RAD23B also functions in DNA damage repair as part of the XPC complex that stimulates the nucleotide excision repair activity. Moreover, RAD23B participates as an adaptor protein in the process of protein degradation. Therefore, the interaction of DDI2 and RAD23B might have important implications for both known functions of DDI2. This work describes the DDI2 and RAD23B interaction on the structural level. Recombinant protein variants of both DDI2 and RAD23B proteins were prepared and the interaction was mapped by the affinity pull-down assay. Protein NMR titrations were further used to explore the interaction. Key words: ubiquitin-proteasome system, DNA damage repair, proteasome expression regulation, aspartyl protease, DDI2, NMR
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Exploring novel strategies targeting HBV
Šmilauerová, Kristýna ; Grantz Šašková, Klára (advisor) ; Černý, Jan (referee)
An effective and safe vaccine against Hepatitis B virus already exists, yet morbidity and mortality of this illness are still high. The key to developing a reliable treatment is a deep knowledge of the virus' life cycle and functions of all its components. In the presented work we explored an interactome of the Core protein of the Hepatitis B virus. Using proximity-dependent biotin identification technique (BioID) coupled to mass spectrometry we have identified a list of potential candidates that are either significantly enriched (in total 105 proteins) or less abundant in the presence of the HBV Core protein in the cell (40 proteins). The list also includes known HBV Core interacting proteins SRPK1 and SRPK2, and p53 protein whose expression is known to be repressed due to the HBV Core interaction with the E2F1 transcription factor. Many of the newly identified possible HBV Core interacting proteins are involved in biological processes already known or are suspected to be influenced by the HBV such as translational and transporting processes or gene expression and macromolecule production. Overall, this work comprehensively characterizes the interaction landscape of the HBV Core protein in the live cells and might thus serve as a reliable start for in depth HBV-host interaction analysis. Key...
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Role of yeast WSS1 protease in DNA repair.
Adámek, Michael ; Grantz Šašková, Klára (advisor) ; Čáp, Michal (referee)
Sustaining the integrity of DNA throughout the lifetime is critical for every living organism. Therefore organisms evolved numerous ways to detect and repair different types of DNA damage caused by various endogenous and exogenous factors resulting in replication stress. Defects in these repair mechanisms can lead to severe human diseases such as neurological disorders, familial cancers or developmental syndromes. In presented master thesis, we investigated the function of a yeast protein named Wss1, a metalloprotease that participates in a recently discovered DNA repair pathway that proteolytically removes DNA-protein crosslinks. Wss1 shows strong negative interaction with another DNA repair protease, Ddi1, in which case was discovered, that double-deleted yeast strain lacking WSS1 and DDI1 is hypersensitive to hydroxyurea. Hydroxurea is a ribonucleotide reductase inhibitor that, in the end, arrests cells in the S-phase of cell-cycle. Based on previous studies, we performed rescue experiments with various deletions and single-site mutants of Wss1p to assess the involvement of particular yeast Wss1p domains in the replication stress response to hudroxyurea.
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Deciphering the biological role of Ddi1-like protein family
Sivá, Monika ; Grantz Šašková, Klára (advisor) ; Bařinka, Cyril (referee) ; Stopka, Pavel (referee)
Ddi1-like protein family has been recently raised into the spotlight by the scientific community due to its important roles in cellular homeostasis maintenance. It represents a specific group among shuttling proteins of the ubiquitin-proteasome system. When compared to other shuttles, Ddi1-like protein family members harbor a unique retroviral-protease like domain besides the conventional ubiquitin-like (UBL) domain and domains interacting with ubiquitin. In addition, a helical domain of Ddi (HDD) has been recently found in most of the orthologs. In this thesis, I focus on characterization of several members of Ddi1-like protein family, both on molecular level using NMR and in model mouse strains via a variety of biological methods. Solution structure of the UBL domain of Ddi1p of S. cerevisiae was solved and its characteristics were compared to those of the UBL domain of its human ortholog. Furthermore, we show that human DDI2 specifically binds to ubiquitin with its terminal domains, both the UBL and the UIM; however, with very low affinity in contrast to binding properties of its yeast counterpart. Our study also show that hDDI2 does not form a head-to-tail homodimer. Based on our structural studies, we hypothesize that human DDI2 might have evolved a different function compared to its yeast...
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Preparation of fusion ligands and evaluation of their binding to NK cell receptors
Nepokojová, Tereza ; Vaněk, Ondřej (advisor) ; Grantz Šašková, Klára (referee)
Natural killer cells (NK cells) are an important part of innate immunity. On their surface they express a complex group of receptors that use different signalling motifs to activate or inhibit NK cell cytotoxic activity. NK cells are capable to kill aberrant cells (namely, viral, infected, and tumour cells) by using special cytotoxic mechanisms to trigger apoptosis. The activating receptors recognize tumour or stress-induced ligands, e.g., NKG2D receptor recognizes the MICA ligand and NKp30 recognizes the B7-H6 ligand. Therefore for human immune system it is only natural that cancer cells are destroyed by NK cells. The current therapeutic goals in the treatment of cancer are primarily focused on strengthening the body's own natural ability to fight with cancer and one possible way is stimulation of NK cells to win this deadly fight. In addition to NK cells, antibodies are also widely used for the treatment of cancer, as well as other immune-related disorders. Most of them are monoclonal antibodies, but antibody fragments are getting attention and are being tested more and more in recent years. This work describes the preparation of three bifunctional fusion proteins: B7-H6-L-aHER2, MICA-L-aHER2, and aHER2-L-MICA, which contain immunoligands for the activating receptors of NK cell and VHH fragment...
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Preparation of nanoparticles for hepatitis B viral therapy
Kružíková, Zuzana ; Grantz Šašková, Klára (advisor) ; Žáčková Suchanová, Jiřina (referee)
Hepatitis B virus (HBV) represents one of the hot topics of current basic and pharmaceutical research. Although an effective vaccine against HBV exists since 1982, the world prevalence of chronic infection is still alarming. The infection can lead to significant liver damage, often resulting in hepatocellular carcinoma. Chronic HBV infection cannot be cured due to the fact that the viral genome persists in the infected hepatocyte hidden from the host immune response as well as from the antiviral treatment. One of the novel approaches aiming for HBV cure suggests that this cccDNA pool could be destroyed using gene editing tools such as CRISPR/Cas9 system. In order to shift this gene editing system to possible medicinal application, CRISPR/Cas9 has to be specifically delivered into the target cell in order to minimize its putative off-target activity. This thesis focuses at first on the design and efficacy testing of new sgRNAs targeting HBV cccDNA and secondly, it describes modular lipid nanoparticles developed specially for delivery of the CRISPR/Cas9 system in the form of RNA. Keywords: hepatitis B virus, CRISPR/Cas9, gene editing, lipid nanoparticles, mRNA delivery, targeted delivery
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